It is known that certain proteins exhibit greater biological activity when attached to other moieties, either by formation of multimeric complexes, where the proteins have an opportunity to act in concert, or through other alterations in the protein""s physico-chemical properties, such as the protein""s absorption, biodistribution and half life. Thus, one current area of research in biotechnology involves the development of methods to modify the physico-chemical properties of proteins so that they can be administered in smaller amounts, with fewer side effects, by new routes, and with less expense.
For example, the binding affinity of any single protein (such as a ligand for its cognate receptor) may be low. However, cells normally express hundreds to thousands of copies of a particular surface receptor, and many receptor-ligand interactions take place simultaneously. When many surface molecules become involved in binding, the total effective affinity is greater than the sum of the binding affinities of the individual molecules. By contrast, when ligand proteins are removed from the cell surface and purified, or isolated by recombinant DNA techniques for use, e.g., as therapeutics, they act as monomers and lose the advantage of acting in concert with many other copies of the same protein associated closely on the surface of a cell. Thus isolated, the low affinity of a protein for its receptor may become a serious drawback to its effectiveness as a therapeutic to block a particular binding pathway, since it must compete against the high avidity cell-cell interactions. Effective treatment might require constant administration and/or high doses. Such drawbacks might be avoided, however, if a means could be found to provide multimeric forms of an isolated protein.
Similarly, it would be useful to modify other physico-chemical properties of biologically active proteins so that, for instance, a protein is induced to associate with a membrane thus localizing it at the site of administration and enhancing its ability to bind to, or otherwise interact with, a particular target. Such changes may also affect the pharmaco-distribution of the protein.
Several methods of generating coupled proteins have been developed. Many of these methods are not highly specific, i.e., they do not direct the point of coupling to any particular site on the protein. As a result, conventional coupling agents may attack functional sites or sterically block active sites, rendering the coupled proteins inactive. Furthermore, the coupled products may be oriented so that the active sites cannot act synergistically, thereby rendering the products no more effective than the monomeric protein alone.
As an additional motivation to find new methods for protein modification, proteins with an N-terminal cysteine residue are susceptible to oxidation or other chemical modifications that may compromise activity or half-life. Additionally, certain buffers commonly used in protein purification have components or impurities that can modify the N-terminal cysteine. Even when these buffers are avoided, the N-terminal cysteine is modified over time, perhaps due to chemicals in the storage vials or in the air. Consequently, formulation buffers must include a protective agent, such as dithiothreitol, to prevent cysteine modification and/or oxidation. However, protective agents have significant biological activity of their own and they may therefore complicate experiments and adversely affect the therapeutic utility of a formulation.
Accordingly, there is a need in the art to develop more specific means to obtain derivatized products or multimeric forms thereof so as to alter the properties of the protein in order to affect its stability, potency, pharmacokinetics, and pharmacodynamics.
In one aspect of the invention, we have solved the problem of finding a way to conveniently make modified forms of biologically active proteins. Methods of the invention can be used to derive multimeric forms of the proteins and/or can be used to change their physico-chemical properties. We have found that modifying a protein (i.e, adding or appending a hydrophobic moiety to an existing amino acid or substituting a hydrophobic moiety for an amino acid) so as to introduce the hydrophobic moiety onto a protein can increase the protein""s biological activity and/or its stability. For example, an N-terminal cysteine can be used as a convenient xe2x80x9ctargetxe2x80x9d to attach a hydrophobic moiety (e.g., a lipid) and thereby modify biologically active proteins.
Alternatively, a hydrophobic moiety can be attached to a C-terminal residue of a biologically active protein, such as hedgehog protein, to modify the protein""s activity. A hydrophobic moiety can also be appended to an internal amino acid residue to enhance the protein""s activity, provided the modification does not affect the activity of the protein, e.g., the proteins ability to bind to a receptor or co-receptor, or affect the protein""s 3-dimensional structure. Preferably, the hydrophobic moiety is appended to an internal amino acid residue that is on the surface of the protein when the protein is in its native form. The hydrophobic modification of the invention provides a generically useful method of creating proteins with altered physico-chemical properties as compared to non-modified forms.
This invention originated, at least in part, from the discovery that when we expressed full-length Sonic hedgehog protein in insect and in mammalian cells, the mature form of the protein (residues 1-174 in the mature sequence), in addition to having cholesterol at the C-terminus, is also derivatized at its N-terminal end with a fatty acid. Significantly, this form of hedgehog exhibited about a 30-fold increase in potency as compared to soluble, unmodified hedgehog in an in vitro assay.
One aspect of the invention is therefore an isolated, protein comprising an N-terminal amino acid and a C-terminal amino acid, wherein the protein is selected from the group consisting of a protein with an N-terminal cysteine that is appended with at least one hydrophobic moiety; a protein with an N-terminal amino acid that is not a cysteine appended with a hydrophobic moiety; and a protein with a hydrophobic moiety substituted for the N-terminal amino acid. The hydrophobic moiety can be a hydrophobic peptide or any lipid or any other chemical moiety that is hydrophobic.
The protein may be modified at its N-terminal amino acid and preferably the N-terminal amino acid is a cysteine or a functional derivative thereof. The protein may be modifed at its C-terminal amino acid or at both the N-terminal amino acid and the C-terminal amino acid, or at at least one amino acid internal to (i.e., intermediate between) the N-terminal and C-terminal amino acids, or various combinations of these configurations. The protein can be an extracellular signaling protein and in preferred embodiments, the protein is a hedgehog protein obtainable from a vertebrate source, most preferably obtainable from a human and includes Sonic, Indian, and Desert hedgehog.
Another embodiment is an isolated, protein of the form: A-Cys-[Sp]-B- X, wherein
A is a hydrophobic moiety;
Cys is a cysteine or functional equivalent thereof;
[Sp] is an optional spacer peptide sequence;
B is a protein comprising a plurality of amino acids, including at least one optional spacer peptide sequence; and
X is optionally another hydrophobic moiety linked to the protein.
The isolated protein can be an extracellular signaling protein, preferably a hedgehog protein. This protein can be modified at at least one other amino acid with at least one hydrophobic moiety. In other embodiments, the protein is in contact with a vesicle selected from the group consisting of a cell membrane, micelle and liposome.
Another aspect of the invention is an isolated, protein having a C-terminal amino acid and an N-terminal thiaproline group, the thiaproline group formed by reacting an aldehyde with, e.g., an N-terminal cysteine of the protein. A further aspect of the invention is isolated, protein having a C-terminal amino acid and an N-terminal amide group, the amide group formed by reacting a fatty acid thioester with an N-terminal cysteine of the protein. Yet another aspect of the invention is an isolated, protein having a C-terminal amino acid and an N-terminal maleimide group, the N-terminal maleimide group formed by reacting a maleimide group with the N-terminal cysteine of the protein. Yet another aspect of the invention is an isolated, protein having a C-terminal amino acid and an N-terminal acetamide group. A further aspect of the invention is an isolated, protein having a C-terminal amino acid and an N-terminal thiomorpholine group.
In these embodiments, the C-terminal amino acid of the protein can be modified with an hydrophobic moiety. The isolated protein can be an extracellular signaling protein, most preferably a hedgehog protein.
Methods of the invention include a method of generating a multivalent protein complex comprising the step of linking, in the presence of a vesicle, a hydrophobic moiety to an N-terminal cysteine of a protein, or a functional equivalent of the N-terminal cysteine. The linking step may include linking a lipid moiety which is selected from saturated and unsaturated fatty acids having between 2 and 24 carbon atoms. The protein can be an extracellular signaling protein, preferably a hedgehog protein selected from the group consisting of Sonic, Indian and Desert hedgehog.
Yet another method of the invention is a method for modifying a physico-chemical property of a protein, comprising introducing at least one hydrophobic moiety to an N-terminal cysteine of the protein or to a functional equivalent of the N-terminal cysteine. The hydrophobic moiety can be a lipid moiety selected from saturated and unsaturated fatty acids having between 2 and 24 carbon atoms. It can also be a hydrophobic protein The protein modified using this method can be an extracellular signaling protein, preferably a hedgehog protein selected from the group consisting of Sonic, Indian and Desert hedgehog. Protein complexes produced by these methods are also encompassed by the present invention.
Other extracellular signaling proteins useful in the invention besides hedgehog include gelsolin; an interferon, an interleukin, tumor necrosis factor, monocyte colony stimulating factor, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, erythropoietin, platelet derived growth factor, growth hormone and insulin.
Another method of the invention for modifying a protein (such as an extracellular signaling protein) that has an N-terminal cysteine. This method comprises reacting the N-terminal cysteine with a fatty acid thioester to form an amide, wherein such modification enhances the protein""s biological activity.
In a preferred embodiment, the fatty acid thioester is an acylated thiophenol, or active thioester, even more preferably, an acylated thiophenol having at least one electron withdrawing substituent on the phenyl ring of the thiophenol. Suitable electron-withdrawing substituents include nitro, carbonyl (e.g., ester, aldehyde, ketone, amide, carboxyl, or thioester groups), cyano, halogen (preferably chloro or fluoro), sulfonyl (e.g., sulfonamide, sulfonate, sulfonic acid, or sulfone groups), or phosphonyl (e.g., phosphate, phosphate, etc.) groups, preferably nitro, carbonyl, cyano, or halogen groups. Two or more such groups may be present on the phenyl ring. In certain embodiments, the thiophenol may be a polycyclic aromatic thiol, e.g., mercaptonaphthalene, mercaptoanthracene, mercaptofluorene, etc., or a heteroaromatic thiol, e.g., mercaptothiophene, mercaptopyridine, mercaptoindole, mercaptoquinoline, etc.
Yet another method is a method for modifying a protein (such as an extracellular signaling protein) having an N-terminal cysteine, which comprising reacting the N-terminal cysteine with a maleimide group, wherein such modification enhances the protein""s biological activity. Other embodiments of this method involve reacting the N-terminal cysteine with either an aldehyde group, an acetamide group or a thiomorpholine group.
A further method is a method for modifying protein (such as an extracellular signaling protein) comprising appending an hydrophobic peptide to the protein. The hydrophobic moiety can be appended to an amino acid of the protein selected from the group consisting of the N-terminal amino acid, the C-terminal amino acid, an amino acid intermediate between the N-terminal amino acid and the C-terminal amino acid, and combinations of the foregoing. In one embodiment, the present invention provides hedgehog polypeptides which are modified with lipophilic moieties. In certain embodiments, the hedgehog proteins of the present invention are modified by a lipophilic moiety or moieties at one or more internal sites of the mature, processed extracellular domain, and may or may not be also derivatized with lipophilic moieties at the N or C-terminal residues of the mature polypeptide. In other embodiments, the polypeptide is modified at the C-terminal residue with a hydrophobic moiety other than a sterol. In still other embodiments, the polypeptide is modified at the N-terminal residue with a cyclic (preferably polycyclic) lipophilic group. Various combinations of the above are also contemplated. A therapeutic method of the invention is a method for treating a neurological disorder in a patient comprising administering to the patient a hydrophobically-modified protein of the invention.